The field of recombinant DNA technology has led to the development of numerous systems for producing a wide variety of naturally occurring and synthetic polypeptides in such microorganisms as yeast and bacteria. Notwithstanding these developments, there is a continuing need to provide for more efficient and economic methods for producing the polypeptides. In developing a process for commercial production of polypeptides, many factors are involved in optimizing the economic and efficient production of the polypeptides. Included among these factors are regulatory signals, which are nucleic acid (DNA and/or RNA) sequences involved with the regulation of gene replication, transcription and translation.
Translation is a multi-stage process which first involves the binding of messenger RNA (mRNA) to ribosomes. Beginning at the translation start codon, the mRNA codons are read sequentially as the ribosomes move along the mRNA molecule. The specified amino acids are then sequentially added to the growing polypeptide chain to yield the protein or polypeptide encoded in the mRNA.
As indicated, the first step in the translation process is the binding of the mRNA molecule to the ribosome. The nature of this interaction (i.e., binding) has been only partially elucidated. Analysis of RNase-resistant oligonucleotides isolated from bacterial translation initiation complexes indicate that a RNA fragment approximately 30 to 40 bases (nucleotides) in length comprises this initial ribosomes binding site. The start codon, which in most cases in an AUG, is located at or around the center of this ribosome binding site. Hence, a ribosome binding site (R.B.S.) is hereinafter understood to comprise a sequence of mRNA surrounding the translation start codon which is responsible for the binding of the ribosome and for initiation of translation.
In most procaryotic ribosome binding sites, the start codon is preceded by a purine rich region at a distance of 5 to 9 bases. The so-called Shine-Dalgarno sequence (Shine-Dalgarno, 1974) shows a variable region of complementarity with a region close to the 3' end of the 16S ribosomal RNA (rRNA). The importance of this region has been demonstrated both directly, by changing this sequence and also indirectly, by comparing the sequences of several known Shine-Dalgarno sequences. Both the Shine-Dalgarno sequence and 16S regions can be co-isolated from initiation complexes as an RNA duplex. The Shine-Dalgarno sequence has thus been found to base-pair with a specific sequence and within a specific region of the bacterial 16S ribosomal RNA. The Shine-Dalgarno region (SD-region) or SD-sequence is thought to assist the 30S particle in positioning itself at the proper place with respect to the start codon on the mRNA. Variation of the distance between the AUG and the SD-region and base composition in this spacer mRNA sequence have been found to affect the efficiency of the translation initiation process. Hence, attempts to optimize the translation efficiency of mRNA in such bacteria as E. coli have thus far centered around three components, the start/signal codon (AUG), the SD-region, and the length and nucleic acid composition of the spacer in between the AUG and SD-region. Other manipulations at the level of translation have included removal of mRNA secondary structure at or around the start of translation and/or substitution of preferred bacterial codons for otherwise less desirable codons in, for example, foreign mRNA. These latter manipulations must be made on an individual mRNA basis and thus do not provide a generic means by which polypeptide production, in general, may be enhanced.
Additionally, the state of the art has not reached a point where high-level expression of foreign gene products in such microorganisms as E. coli is a routine and predictable operation. The term "foreign" as used herein means genes, proteins and/or nucleic acid molecules not normally present within the specified host cell. Subtle features of the foreign gene, mRNA and protein can all affect the expression machinery of the microorganism leading to reduced accumulation of the desired product. Specifically, the efficiency of expression of known procaryotic genes varies by a factor of around 1,000 (Gold et al. 1984).
To achieve high levels of gene expression in such procaryotic hosts as E. coli, it is necessary to use not only strong transcriptional promoters to generate large quantities of mRNA, but also to identify a ribosome binding site(s) that ensure that the mRNA is efficiently translated. There is, therefore, a need to create a binding site which correlates with a predictable increased or enhanced level of translation for a wide variety of genes (e.g. procaryotic and eucaryotic).
In one publication by Gold et al. (1984), a non-SD-region in an E. coli mRNA able to base-pair with the E. coli 16S ribosomal RNA was reported. However, said mRNA molecule was also found to contain a novel translation initiation codon, AUU, which codon is unique among E. coli mRNAs. (Gold et al. 1984).